The Dynamic Accumulation Rules of Chemical Components in Different Medicinal Parts of Angelica sinensis by GC-MS

The chemical components and medicinal properties of different medicinal parts of Angelica sinensis are often used as medicine after being divided into the head, body and tail of Angelica sinensis. In this study, the chemical components of different medicinal parts in different periods were analyzed by GC-MS for the first time, and the differences of the accumulation rules of chemical components in different medicinal parts of Angelica sinensis were obtained. This study demonstrated that the differences of composition accumulation in different medicinal parts of Angelica sinensis were mainly reflected in the types and relative contents of compounds. The study found that the number of compounds in different medicinal parts of Angelica sinensis in each period were different and the change rules of the same compound in different medicinal parts were also different. The number of compounds in the tail of Angelica sinensis was the least in April, and the largest in October. The content of ligustilide in the body of Angelica sinensis was higher in April and was the highest in the tail in October. The relative content of butylidenephthalide in the head was the highest in October. The relative contents of senkyunolide A and butylphthalide in the head were decreased in October, while the contents in the body and tail increased, indicating that the compounds that accumulate in the head may transfer to the body and tail in later stages of growth. This study clarified the differences in the accumulation of chemical components in different medicinal parts of Angelica sinensis, which could provide a theoretical basis for the reasons for the differences of chemical components in the different medicinal parts.


Introduction
Angelica sinensis Radix (A. sinensis) is the dried root of the Angelica Sinensis (Oliv.) Diels [1]. It has the effect of replenishing blood, activating blood circulation and regulating irregular menstruation [2,3]. During the Ming and Qing Dynasties, doctors clearly divided A. sinensis into head (the upper part of the root), body (the taproot) and tail (lateral roots). It is divided into different parts and used as medicine. The main function of the head is to stop bleeding, the body has a strong effect of replenishing blood and the tail focuses on promoting blood circulation and removing blood stasis. There is a high correlation between the difference in the medicinal properties of the different parts of A. sinensis and the basis of the chemical substances [4]. Studies have found that the content of the same substance in the different medicinal parts was different, the content of volatile oil and ferulic acid in the tail of A. sinensis was the highest, and the content of total tannins in head was the highest [5]. Other researchers determined the content of ferulic acid in different medicinal parts of A. sinensis by HPLC and also reached the conclusion that the content of ferulic acid in the tail of A. sinensis was the highest [6].
The accumulation dynamics of chemical components in medicinal plants is closely related to growth and development. A. sinensis is a perennial herb with a total growth

GC-MS Column
The column used was a DB-23 (30 m × 0.25 mm × 0.25 μm). The carrier gas was high purity helium, and its flow rate was 1.0 mL/min. The initial temperature was maintained at 60 °C for 3 min, and then raised to 270 °C at a rate of 10 °C/min. All samples were injected in split mode at 270 °C. The split injection was 5:1.

Mass Spectrum Conditions
An electron impact ion source was used, with full scanning mode (mass range m/z 50-650), ion source temperature 230 °C, interface temperature 250 °C, quadrupole temperature 150 °C, electronic energy 70 eV and solvent delay time 3 min. The ion detection mode selected ion monitor was selected.

Data Analysis
GC-MS was used to perform a full ion scan of the compound, and the total ion current map of different medicinal parts of A. sinensis in different periods was obtained. The compound was qualitatively analyzed by searching the NIST14 standard mass spectrometry library, and according to the peak area normalization method the relative content of each component was calculated (see Supplementary Materials Table S1).

Analysis of Chemical Components in the Head of A. sinensis in Different Periods
The sample solution of the head of A. sinensis in different periods was analyzed by GC-MS. The GC-MS chromatogram in different periods was shown in Figure 2. The chemical composition and relative concentrations were obtained using the peak area normalization method (Table 1).

Mass Spectrum Conditions
An electron impact ion source was used, with full scanning mode (mass range m/z 50-650), ion source temperature 230 • C, interface temperature 250 • C, quadrupole temperature 150 • C, electronic energy 70 eV and solvent delay time 3 min. The ion detection mode selected ion monitor was selected.

Data Analysis
GC-MS was used to perform a full ion scan of the compound, and the total ion current map of different medicinal parts of A. sinensis in different periods was obtained. The compound was qualitatively analyzed by searching the NIST14 standard mass spectrometry library, and according to the peak area normalization method the relative content of each component was calculated.

Analysis of Chemical Components in the Head of A. sinensis in Different Periods
The sample solution of the head of A. sinensis in different periods was analyzed by GC-MS. The GC-MS chromatogram in different periods was shown in Figure 2. The chemical composition and relative concentrations were obtained using the peak area normalization method (Table 1).
The relative contents of the compounds in different periods were analyzed by the peak area normalization method, and the accumulation dynamics of four important active components (see the right photo of Figure 1) in the head of A. sinensis were analyzed. As shown in Figure 3, the relative content of (Z)-ligustilide increased gradually from April to October and increased rapidly from September to October. The relative content of (Z)-3-butylidenephthalide decreased by 0.0082% in June, and then showed a gradual upward trend. The relative contents of senkyunolide A and 3-butylisobenzofuran-1(3H)one reached the maximum value of 3.6762% and 0.7921% in September, and the contents decreased in the harvest period. Combining the changes of compounds in each period, (Z)-ligustilide was the highest content component from seedling stage to harvest stage, and the relative content was up to 72.2466% in harvest stage, which was the main core component in the head of A. sinensis in each period.
Molecules 2022, 27, x FOR PEER REVIEW 6 of 15 ligustilide was the highest content component from seedling stage to harvest stage, and the relative content was up to 72.2466% in harvest stage, which was the main core component in the head of A. sinensis in each period.

Analysis of Chemical Components in the Body of A. sinensis at Different Periods
The GC-MS chromatogram of A. sinensis of the body of A. sinensis in different periods was shown in Figure 4. The chemical composition and relative concentrations were obtained using the peak area normalization method ( Table 2). The types of chemical components in the body of A. sinensis varied greatly in different periods.

Analysis of Chemical Components in the Body of A. sinensis at Different Periods
The GC-MS chromatogram of A. sinensis of the body of A. sinensis in different periods was shown in Figure 4. The chemical composition and relative concentrations were obtained using the peak area normalization method ( Table 2). The types of chemical components in the body of A. sinensis varied greatly in different periods.

Analysis of Chemical Components in the Body of A. sinensis at Different Periods
The GC-MS chromatogram of A. sinensis of the body of A. sinensis in different periods was shown in Figure 4. The chemical composition and relative concentrations were obtained using the peak area normalization method ( Table 2). The types of chemical components in the body of A. sinensis varied greatly in different periods. In April, the compounds in the body were the same as those in the head of A. sinensis. They mainly contain Z-ligustilide, (Z)-3-butylidenephthalide, senkyunolide A and other ester compounds and a small amount of olefin compounds. In June, 12 components were added, and 4 compounds were added compared with the head in the same period: 1,2,6,6tetramethylcyclohexa-1,3-diene, α-acorenol and trans-Isoeugenol. A total of 48 components were identified in the body of A. sinensis in August. In addition to a large number   As shown in Figure 5, it was found that from April to June, the contents of Z-ligustilide, (Z)-3-butylidenephthalide and senkyunolide A in the body of A. sinensis decreased by 0.2584%, 0.0235% and 0.0183%, 3-butylisobenzofuran-1(3H)-one increased by 0.0092%. The aboveground part grew vigorously in June, and these three reduced compounds might be involved in the transformation of the compounds when the aboveground part grew. The content of senkyunolide A decreased by 0.0525% in September; the other three compounds showed an increasing trend from June to October, entered a rapid accumulation period in September and reached the maximum value in October. The relative contents of Z-ligustilide, (Z)-3-butylidenephthalide, senkyunolide A and 3-butylisobenzofuran-1(3H)one were: 71.3681%, 0.9806%, 1.5863% and 0.5095%.

Analysis of Chemical Components in the Tail of A. sinensis at Different Periods
The GC-MS chromatogram of A. sinensis of the tail of A. sinensis in different periods was shown in Figure 6. The chemical composition and relative concentrations were obtained using the peak area normalization method (Table 3).

Analysis of Chemical Components in the Tail of A. sinensis at Different Periods
The GC-MS chromatogram of A. sinensis of the tail of A. sinensis in different periods was shown in Figure 6. The chemical composition and relative concentrations were obtained using the peak area normalization method (Table 3).
As shown in Figure 7, in the tail of A. sinensis, Z-ligustilide, (Z)-3-butylidenephthalide, senkyunolide A and 3-butylisobenzofuran-1(3H)-one were also selected to analyze the relative content change trend. The results showed that the contents of these four compounds showed a gradual upward trend from April to October. The growth was relatively slow from April to June, entered the period of rapid growth after June, and reached the highest value in October. The relative contents were 73.4925%, 0.8135%, 1.4591% and 0.3314%, respectively.

Analysis of the Differences in the Accumulation of Compounds in Different Medicinal Parts of A. sinensis
The number of compounds identified in different medicinal parts of A. sinensis in different periods was shown in Table 4. From April to October, the number of compounds in the head, body and tail of A. sinensis showed an increasing trend. In April, there was one less compound in the tail than in the head and body. In June, there were 11 and 7

Analysis of the Differences in the Accumulation of Compounds in Different Medicinal Parts of A. sinensis
The number of compounds identified in different medicinal parts of A. sinensis in different periods was shown in Table 4. From April to October, the number of compounds in the head, body and tail of A. sinensis showed an increasing trend. In April, there was one less compound in the tail than in the head and body. In June, there were 11 and 7 more compounds in tail than head and body, respectively. It mainly contained olefin compounds such as β-chamigrene and 1,2,6,6-tetramethylcyclohexa-1,3-diene and ester compounds such as (Z)-9-octadecenoic acid, methyl ester and 7,10-octadecadienoic acid, etc. Since then, the species of compounds in the tail of A. sinensis were always the highest. During the harvest period, the number of species gradually reached a balance, and the number of compounds in head, body and tail were 61, 68 and 69, respectively, and there were differences in the types of components. In the harvest period, 6-epi-shyobunol only existed in the head of A. sinensis, eicosapentaenoic acid was found only in the body of A. sinensis, and 10,13-Octadecadiynoic acid and α-Elemen were only found in the tail of A. sinensis. 9-Hexadecenoic acid, 9-Octadecen-12-ynoic acid methyl ester, limonen-6-ol, pivalate, (+)-cuparene, carveol and (Z,Z)-9,12-octadecadienoic acid were not found in the head but existed in body and tail of A. sinensis. The relative content changes of the four main active components in A. sinensis in different medicinal parts were shown in Figure 8. In April, the content of Z-ligustilide in the body of A. sinensis was the highest, 0.8323% higher than that in the tail. From June, the content of Z-ligustilide in the tail was gradually higher than that in the head and body. From June to October, the relative content remained as tail > head > body. During the rapid accumulation of content in September, the difference of relative content in head, body and tail gradually decreased and tended to balance.  As one of the important active components in A. sinensis, the accumulation law of (Z)-3-butylidenephthalide was different from that of Z-ligustilide. In April, the relative content of (Z)-3-butylidenephthalide in the head was higher, which was 0.0159% higher than that in the tail. In August, the content in the head was higher, and from August to As one of the important active components in A. sinensis, the accumulation law of (Z)-3-butylidenephthalide was different from that of Z-ligustilide. In April, the relative content of (Z)-3-butylidenephthalide in the head was higher, which was 0.0159% higher than that in the tail. In August, the content in the head was higher, and from August to October, the relative content gap between the head and the body and the tail gradually increased, and the highest content in the head was 1.3462% during the harvest period, which was 0.5327% higher than that in the tail. Researchers [17] analyzed the chemical components of different parts by GC-MS and found that the relative contents of compounds in different parts were different. Z-ligustilide was the main compound, which was consistent with the results of our study. Ligustilide compounds affect platelet aggregation or thrombosis [18], and the content of Z-ligustilide in tail was the highest, which was consistent with the effect of tail focusing on promoting blood circulation.
In April, the content of senkyunolide A in the body was higher; the content in the head had always been the highest since August and reached the highest value of 3.6762% in September. In October, the content in the head of A. sinensis decreased by 2.0498%, while the content in the body and tail increased by 1.2184% and 0.3708%, respectively, which may be caused by the transfer of senkyunolide A from head to body and tail.
From April to September, the content of 3-Butylisobenzofuran-1(3H)-one in the head was always the highest and decreased by 0.3047% in the head and increased by 0.3046% and 0.0477% in the tail by the harvest period. The relationship of the content of 3-Butylisobenzofuran-1(3H)-one at the harvest period was: body > head > tail. In September, the aboveground parts withered gradually, the required nutrients decreased gradually and a large number of assimilates transferred to the roots. At this time, the contents of (Z)-3-butylidenephthalide, senkyunolide A and 3-butylisobenzofuran-1(3H)-one decreased in the head and increased in the body and tail, which can confirm this inference. It was speculated that there was a phenomenon that the compounds that accumulated in the head transferred to the body and tail during the accumulation of effective components in different medicinal parts. Overall, the accumulation and distribution of compounds were consistent with the efficacy of different medicinal parts of A. sinensis and the accumulation of the types and contents of the compounds reached the maximum in October, which was consistent with the traditional harvesting period [19].
The content changes of the same compound in different medicinal parts of A. sinensis in different periods may be caused by some related factors during growth and development such as environmental factors, temperature and solar radiation, which would accelerate the synthesis of a compound, thus slowing down the synthesis of other related compounds [20,21]. But the specific mechanism is not clear at present.

Conclusions
In summary, we revealed the differences of accumulation dynamics in different medicinal parts of A. sinensis by GC-MS for the first time, which were mainly reflected in the component types and relative contents. The number of compounds contained in different medicinal parts of A. sinensis showed an increasing trend from April to October, but the number of compounds in different medicinal parts was different in each period and the types of components were also different. This study revealed the accumulation rule of chemical constituents in different medicinal parts of A. sinensis and provided a theoretical basis for the differences in compounds and medicinal properties in different medicinal parts.